WO2007016254A2 - Activites antineoplasiques de l'ellipticine et de ses derives - Google Patents

Activites antineoplasiques de l'ellipticine et de ses derives Download PDF

Info

Publication number
WO2007016254A2
WO2007016254A2 PCT/US2006/029212 US2006029212W WO2007016254A2 WO 2007016254 A2 WO2007016254 A2 WO 2007016254A2 US 2006029212 W US2006029212 W US 2006029212W WO 2007016254 A2 WO2007016254 A2 WO 2007016254A2
Authority
WO
WIPO (PCT)
Prior art keywords
ellipticine
myeloma
cells
cell
eped3
Prior art date
Application number
PCT/US2006/029212
Other languages
English (en)
Other versions
WO2007016254A3 (fr
Inventor
John Shaughnessy
Erming Tian
Original Assignee
The Board Of Trustees Of The University Of Arkansas
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Board Of Trustees Of The University Of Arkansas filed Critical The Board Of Trustees Of The University Of Arkansas
Publication of WO2007016254A2 publication Critical patent/WO2007016254A2/fr
Publication of WO2007016254A3 publication Critical patent/WO2007016254A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates generally to the field of cancer therapeutics. More specifically, the present invention relates to the derivatives of the plant alkaloid, ellipticine, as new therapeutic agents for treating cancer.
  • Ellipticine (5,11-Dimethyl-6H-Pyridol[4,3]carbazole, MW+246.3), an alkaloid isolated from Apocyanaceae plants, is a topoisomerase II inhibitor that induces topo II dependent DNA cleavage. Ellipticine has been shown to exhibit-significant anti-tumor and anti-HIV activity (Stiborova et al., 2001). The antineoplastic mechanism of ellipticine comprised formation of covalent DNA adducts, which was mediated by human cytochrome P450 (CYP). Additionally, the same study also elucidated the metabolites responsible for DNA binding (Stiborova et al., 2004).
  • DTP Developmental Therapeutics Program
  • NCI National Cancer Institute
  • CKSlB which lies within this locus and controls several aspects of cell cycle progression, was significantly related to the prognoses of myeloma in these patients (Shaugnessy, 2005). Despite this, there is lack of therapeutics that target cells with high levels of CKSlB expression.
  • prior art is deficient in drugs target cells that express high levels of CKSlB.
  • the prior art is deficient in effective treatment ⁇ f- for myeloma.
  • the present invention fulfills this long-standing need and desire in the art.
  • the present invention is directed to a method of treating myeloma in an individual. Such a method comprises administering a pharmcologically effective dose of ellipticine, a derivative of ellipticine or a combination thereof.
  • the present invention is also directed to a method of treating myeloma in an individual.
  • a method comprises administering a pharmacologically effective dose of a topoisomerase II inhbitior, where the inhibitor induces cell cyle arrest of myeloma cells, induces apoptosis of myeloma cell, overcomes acquired drug resistance or a combination thereof without affecting the viability of normal cell. This results in treatment of myeloma in the individual.
  • the present invention is further directed to a method of inhibiting growth of a myeloma cell.
  • a method of inhibiting growth of a myeloma cell comprises contacting the myeloma cell with ellipticine, a derivative thereof or a combination thereof.
  • the present invention is still further directed to a method of inducing apoptosis of a myeloma cell.
  • a method of inducing apoptosis of a myeloma cell comprises contacting the myeloma cell with ellipticine, a derivative thereof or a combination thereof such that said contact activates caspase 9, thereby inducing apoptosis of the myeloma cell.
  • FIG. 1 shows the anti-myeloma activity of the four ellipticine derivatives, 19942 J/4 (A), 130789 U/l (X ) 5 316458 K/4 (#), 338258 G/l ( ⁇ ) and 630740 U/l (I) for the myeloma cell line Ark.
  • the dosage of each derivative was set at 0.5 mg/ml.
  • a cell culture medium control (•) and 0.5 % DMSO control (I) was maintained in the experiment.
  • the cell viability was tested each day over a period of five days using the CellTiter-Glo luminescent cell viability assay kit from Promega Co.
  • Figure 2 shows the anti-myeloma activity of the four ellipticine derivatives, 19942 J/4 (A), 130789 U/l (X ), 316458 K/4 (*), 338258 G/l ( ⁇ ) and 630740 U/l (I) for the myeloma cell line kms 11.
  • the dosage of each derivative was set at 0.5 mg/ml.
  • a cell culture medium control (•) and 0.5 % DMSO control (I) was maintained in the experiment.
  • the cell viability was tested each day over a period of five days using the CellTiter-Glo luminescent cell viability assay kit from Promega Co.
  • Figure 3 shows the anti-myeloma activity of the four ellipticine derivatives, 19942 J/4 (A), 130789 U/l (X ), 316458 K/4 (*), 338258 G/l ( ⁇ ) and 630740 U/l (I) for the myeloma cell line RPMI8226.
  • the dosage of each derivative was set at 0.5 mg/ml.
  • a cell culture medium control (•) and 0.5 % DMSO control (I) was maintained in the experiment.
  • the cell viability was tested each day over a period of five days using the CellTiter-Glo luminescent cell viability assay kit from Promega Co.
  • Figure 4 shows the anti-myeloma activity of the four ellipticine derivatives, 19942 J/4 (A), 130789 U/l (X ), 316458 K/4 (#), 338258 G/l ( ⁇ ) and 630740 U/l (I) for the myeloma cell line U266.
  • the dosage of each derivative was set at 0.5 mg/ml.
  • a cell culture medium control (•) and 0.5 % DMSO control (I) was maintained in the experiment.
  • the cell viability was tested each day over a period of five days using the CellTiter-Glo luminescent cell viability assay kit from Promega Co.
  • Figure 5 shows the invitro comparison of ellipticine (EPE), NSC 69187 (EPE-
  • NSC 338258 (EPE-Der3) with respect to their anti-myeloma activity for the four myeloma cell lines, ARK, CAG, RPMl 8226 and U266.
  • the dosage of each derivative was set at 2, 0.2 and 0.02 mM.
  • a cell culture medium control and 0.5 % DMSO control was maintained in each experiment.
  • the cell viability was tested each day over a period of five days using the CellTiter-Glo luminescent cell viability assay kit from Promega Co.
  • Figure 6 shows the comparison of ellipticine analog NSC 338258 (A) with existing anti-myeloma drugs, Adriamycin® ( ⁇ ) and Etoposide ( ⁇ ).
  • a cell culture medium control and 0.5 % DMSO control was maintained in each experiment. The results obtained from these controls are pooled together in the figure (•).
  • the dose of each compound was set at 0.2 mM.
  • the cell viability was tested each day over a period of five days using the CellTiter-Glo luminescent cell viability assay kit from Promega Co.
  • Figure 7 shows the viability of normal fetal bone mesenchymal cells (FB MSC) when contacted with NSC 338258 (EPE-Der3) in the concentration range of 0.01 - 1.0 mM. The cell viability was tested each day over a period of five days using the CellTiter-Glo luminescent cell viability assay kit from Promega Co.
  • Figure 8A shows the overall cell growth inhibition by NSC 388258.
  • Five non myeloma cancer cell lines, HEL (I) 5 HL-60 (A), k562 (X ), MEGOl (#) and THPl ( ⁇ ) were exposed to NSC 388258 over a period of 5 days.
  • the dose was set at 0.2 mM.
  • a cell culture medium control and 0.5 % DMSO control was maintained in each experiment. The results obtained from these controls are pooled together in the figure (•).
  • the cell viability was tested each day over a period of five days using the CellTiter-Glo luminescent cell viability assay kit from Promega Co.
  • Figure 8B shows the overall cell growth inhibition by NSC 388258.
  • Figure 9 shows the chemical structures of ellipticine and EPED3.
  • NSC 338258 (EPED3, right) is a water-soluble derivative of ellipticine with the modification at position 9.
  • Figures 10A-10B show that EPED3 has high anti-myeloma efficacy.
  • U266 cell line was one of 12 myeloma cell lines used in screening of anti-myeloma agents.
  • Figure 1OA cells were treated with 14 ellipticine derivatives (0.5 mM) obtained from the DTP; control cells were untreated.
  • Ellipticine derivatives are coded with NSC identifiers. For 5 days, cell viability was measured every 24 hours using CellTiter-Glo Luminescent Assay.
  • EPED3 exhibits drastic cytotoxic activity on U266 cells.
  • U266 cells along with six other myeloma cell lines, were co-cultured in direct contact with human fetal bone mesenchymal cells and treated with EPED3 (0.5 mM and 2 mM) or Velcade (0.5 mM); control cells were untreated. Cell proliferation was assessed by MTT assay every 24 hours for 5 days. At 2 mM, EPED3 exhibited similar reductions in cell proliferation efficacy as 0.5 mM Velcade. Error bars indicate standard deviation.
  • FIGS 11A-11F compare the morphological features of EPED3 and Velcade induced apoptosis in myeloma cells and normal mesenchymal cells.
  • JJN3 myeloma cell line and human fetal bone mesenchymal cells were co-cultured without direct contact between cell types.
  • EPED3 (2 mM) and Velcade (0.5 mM) were added to the cultures; control cells were untreated.
  • Live cell cultures were photographed 3 days after initiating treatment (40Ox magnification under Olympus invert-microscope with RT-Color SPOT digital camera and the software, Diagnostic Instrument Inc. Sterling Heights, MI).
  • Figures 12A-12B show dose response of myeloma cells to common anti- myeloma agents used in clinics.
  • U266 was one of 4 myeloma cell lines exposed to a panel of anti-myeloma agents in 2-fold titration (6.4-0.1 mM). MTT assays were performed to assess cell proliferation inhibition for 24 hours ( Figure 12A) and 48 hours ( Figure 12B). Without co-culture condition, Velcade exhibits a great efficiency on killing U266 cells (less than 0.1 ⁇ M).
  • EPED3 stopped cell proliferation at 0.2 ⁇ M, and exhibited better efficacy on reduction of cell metabolic activity at 0.4 ⁇ M or greater. Error bars indicate standard deviation.
  • Figures 13A-13B show dose response of RPMI 8226 and 8226/DoxlV myeloma cells to EPED3 ( Figure 13A) or Dox ( Figure 13B).
  • Cells were incubated for 96 hours with drug concentrations as indicated and analyzed by MTT assays; linear regression analysis was used to determine the IC 50 for each drug (EPED3, Figure 13 A; Dox, Figure 13B) and each cell line (RPMI 8226, squares; 8226/Doxl V, circles). Data are presented as the mean of four independent experiments.
  • Percent cells with fragmented DNA are indicated as c% in each graph.
  • Cells were also analyzed for apoptotic status ( Figures 14G-14L) using Coulter Annexin-V FITC/7-AAD kit. Untreated cells (Control; Figures 14G and 14J) were maintained at high viability (90%). Early apoptotic cells were gated at G4, and necrotic cells were gated at G2; percent cells in each population are indicated with each graph. The arrows indicate percent viable cells ingested EPED3, whose fluorescent property was captured at channel FL4 ( Figures 14H, 14K).
  • Figures 15A-15C show fluorescent images of EPED3 endoplasmic distribution and induction of apoptosis, which was also detected by Western Blot.
  • U266 cells (10 6 cells/ml) were co-cultured without contact with human fetal bone mesenchymal cells and treated with EPED3 (2 mM) or Velcade (0.5 mM).
  • EPED3 (2 mM) or Velcade (0.5 mM).
  • nuclei were stained with DAPI (blue), mitochondria with MitoTracker Red CMXRos (red), and cytochrome c with FITC-conjugated secondary antibodies (green);
  • EPED3 is excited by ultraviolet light (wavelength ⁇ 500 nm) and was visualized as gold.
  • Figure 15A shows cells photographed under fluorescence microscopy at the time of treatment and 30 minutes, 1.5 hours, and 6 hours thereafter. The appearance of large cytoplasmic vacuoles (arrows) and diminished mitochondria in cells treated with EPED3 indicate ongoing apoptosis.
  • Figure 15B shows cells treated with EPED3 and Velcade were examined after 6 hours for lyses of mitochondria and release of cytochrome c (green). Cytochrome c disassociation with mitochondria also indicates initiation of the intrinsic apoptotic pathway.
  • Figure 15C compares the expression of caspases in myeloma cell lines (JJN3, L363, OPM2, and U266) were co-cultured without contact with human fetal bone mesenchymal cells and treated with EPED3 (2 mM) or Velcade (0.5 mM) for 6 hours. Protein extracts (100 mg) were analyzed by Western blotting, using goat anti-human Caspase-3, -8, and -9 polyclonal antibodies . Inactivation of Caspase-8 indicates that the extrinsic apoptotic pathway was not induced by EPED3 or Velcade treatments.
  • the present invention is directed to the use of ellipticine and its derivatives in the treatment of cancer cells such as myeloma cells. It has been shown herein that the ellipticine derivatives, for instance NSC 338258 (EPED3) and NSC 69178 were cytotoxic to myeloma cell lines. These ellipticine derivatives were identified as strong inhibitors of cell growth with the effective invitro dosage of 0.2 ⁇ M. Additionally, further comparison of the cell growth inhibitory effects showed NSC 33828 to be more toxic to all cell lines among all other compounds that were tested. Results summarized in Table 1 show that both the ellipticine derivatives have better anti-myeloma activity compared to ellipticine.
  • NSC 338258 was better than NSC 69187 with respect to anti-myeloma activity. These results indicate that ellipticine, NSC 69187 and NSC 338258 can be used in the treatment of myeloma.
  • Table 1 shows the cell viability over a period of five days as determined by the CellTiter-Glo luminescent assay kit from Promega Co.
  • the present invention also demonstrates that the ellipticine derivatives were cytotoxic without possible collateral effects on normal cells.
  • EPED3 the effect of EPED3 on the cell growth of fetal bone derived normal mesenchymal cell was examined. The cell viability was tested every day for a period of five days. It was observed that these cells were not killed by the concentrations that were otherwise toxic to the myeloma cells.
  • Etoposide is an antitumor agent that complexes with topoisomerase II and DNA to enhance double-stranded and single-stranded cleavage of DNA and reversibly inhibit religation. It blocks the cell-cycle in S-phase and G2 phase, induces apoptosis in normal and tumor cell lines, inhibits synthesis of the oncoprotein, Mdm2 and induces apoptosis in tumor lines that overexpress Mdm2.
  • Adriamycin® is a cytotoxic, anthracycline antibiotic used in antimitotic chemotherapy.
  • neoplastic diseases such as acute lymphoblastic leukemia, Wilms' tumor, soft tissue and osteogenic sarcomas, Hodgkin's disease, non-Hodgkin's lymphomas, Ewing's sarcoma and bronchogenic, genitourinary, breast and thyroid carcinoma (IARC 1976). Both these drugs are also used to treat myeloma. It was observed that the toxic effect of EPED3 was immediate compared to the other known anti-neoplastic agents.
  • EPED3 was effective against non-myeloma cell lines such as HEL, HL- 60, k562, MEGOl, THPl, HeIa, g401, dul45, sw480, sw620, saoS2 and MG63.
  • EPED3 was a potent cell growth inhibitor.
  • the preliminary data discussed supra demonstrated that the Ellipticine derivatives were potent inhibitors of myeloma and non-myeloma cancer cell growth. However, these derivatives did not affect the growth of normal cells.
  • EPED3 inhibited the growth of myeloma cells much earlier than the anti-neoplastic agents that were tested.
  • these Ellipticine derivatives could be used to inhibit the growth of myeloma and no- myeloma cancer cells.
  • myeloma cell lines were initially cultivated from peripheral blood (e.g., L363, LP- 1 , OPM2, RPM 8226, U266) or pleural effusion (e.g., ARK 5 ARP 1 , CAG, EJM, H929), all encode the common genetic mutations that exist in primary myeloma.
  • peripheral blood e.g., L363, LP- 1 , OPM2, RPM 8226, U266
  • pleural effusion e.g., ARK 5 ARP 1 , CAG, EJM, H929
  • NCI's DTP has used a panel of 60 human tumor cell lines (NCI-60) derived from various tissues to screen potential anticancer agents for their ability to inhibit growth of multiple cancer cell lines (Monks et al., 1991).
  • NCI-60 human tumor cell lines
  • researchers with similar mechanisms had similar patterns of sensitivity (i.e., some cells are more sensitive to topoisomerase poisons, others less so).
  • COMPARE algorithm (Paull et al., 1989), which compiled a list of compounds with patterns of growth inhibition similar to that of a "seed” compound supplied by an investigator. This approach can suggest potential mechanisms of action for novel compounds.
  • the NCI-60 panel has now been molecularly characterized (Holbeck, 2004; Sausville and Holbeck., 2004), which enables the COMPARE analysis to be extended to gene expression signatures.
  • the bone marrow microenvironment augments myelomagenesis and can protect myeloma cells from destruction when patients undergo systemic and supportive therapies.
  • myeloma cells are reportedly well protected from chemotherapeutic agents (Nefedova et al., 2001).
  • low-dose Velcade (4 nM) is a very effective anti- myeloma agent in the absence of stromal cells; however, when treating myeloma cells in co-culture with human fetal bone mesenchymal cells, myeloma cells exhibit excessive tolerance to Velcade (up to 500 nM), demonstrating more than 100-fold reduction in sensitivity (data not shown).
  • the myeloma cell-mesenchymal cell co-culture system was used in the systematic screening of new synthetic compounds capable of overcoming the factors present in the bone marrow microenvironment that confound therapeutic efficacy.
  • EPED3 was formed by modifying position 9 of ellipticine with a substitute radical of dimethyl amino-ethoxy.
  • the radical preoccupies the hydroxylation position (9) of ellipticine (Chadwick ey al., 1978), which is likely to prevent its further metabolism and tremendously improved hydrophilicity of the compound.
  • EPED3 retained ellipticine' s high fluorescence spectral property (Sureau et al., 1993), absorbing ultraviolet energy at wavelengths 350 nm- ⁇ -95 nm and emitting at 660 nm as weak R-Phycoerythrin-Cyanine 5 signal.
  • EPED3 was located in the mitochondrial inner membrane; this would provide an opportunity to disrupt the membrane potential by uncoupling mitochondrial oxidative phosphorylation, presumably through inhibition of the electron pathway of cytochrome c oxidase.
  • DNA-specific dye such as 7-Amino-actinomycin D (7 -AAD)
  • 7 -AAD 7-Amino-actinomycin D
  • Live cells remained 7-AAD nega tive, apoptotic become 7-AADdim, and late apoptotic or necrotic cells appear 7-AADbright ( Figure 14).
  • the increase of Go+Gt-phase myeloma cells was another mark of cell growth arrest.
  • the present invention used myeloma cell line 8226/Doxl V, a drug-resistant variant of RPMI 8226 cells that is characterized by reduced expression and activity of topo II, which confers insensitivity to a variety of topo II- dependent cytotoxicity. While initially categorized as a topo II inhibitor, ellipticine has now been shown to localize in cytoplasm and accumulate in mitochondria (Chadwick et al.. 1978). The studies of EPED3 cytotoxicity indicated that it, too, was unlikely to directly affect topo II function in myeloma cells.
  • mitochondrial disruption appeared to be the result of instant impact by intracellular EPED3, and that was associated with immediate release of cytochrome c.
  • Cell growth inhibition by DNA intercalation and stimulation of topo II-mediated DNA breakage was a protracted response even in cultured cells.
  • the 8226/Doxl V cells exhibited a high tolerance to Dox ( Figure 13), which functions through topo Il-inhibition, but EPED3 treatment resulted in prompt, dose-dependent killing of myeloma cells.
  • Celiptium (NSC 264137) and Detalliptinium (NSC 311152) are two ellipticine derivatives clinically administrated against breast cancer. Both ellipticines were tested, in vivo, for DNA cleavage activity in comparison to m- AMSA, Amsacrine - a putative topo II inhibitor. Although IC 50 dosages of those agents are in a close range, Celiptium and Detalliptinium were, however, showing 50 times less potent in DNA strand breakage than m-AMSA (Multon et al., 1989). This was in agreement with finding presented herein of lack of topo II-dependent function among ellipticine- derivatives.
  • EPED3 could overcome the acquired drug tolerance to Velcade in myeloma cells.
  • the in vitro chronic exposure to Velcade an inhibitor to proteasome function by targeting the chymotryptic-like site of the molecule (Lee and Gottesman, 1998), has elevated the tolerance level (> 5 nM) to the drug in several myeloma cell lines. It was observed that such Velcade-tolerance did not apply to EPED3, while the cell death occurred at the same low dose of EPED3 as the treatments to those parental myeloma cells (data not shown).
  • EPED3 was given to P388 Leukemia-bearing CD2F1 (CDFl) mice (maximal dose, 200 mg/kg body weight) by intraperitoneal injection. At 50 mg/kg, five out of six animals survived EPED3 toxicity of for at least 5 days. The dosage was equal to approximately 4.5 mM. This preliminary study supports the promise of EPED3 as a new generation of anti-myeloma agents for future preclinical studies to develop tailored therapies for patients with myeloma.
  • the present invention used NCI's COMPARE algorithm to identify ellipticines and several other synthetic compounds that showed a strong correlation between their GI50 for 60 human tumor cell lines (NCI-60) and the cells' expression levels of CKSiB.
  • NCI-60 human tumor cell lines
  • CKSiB CKSiB
  • EPED3 was observed to be highly effective in killing myeloma cells.
  • EPED3 is a highly hydrophilic derivative of ellipticine (figure 9), which is a hydrophobiccell-permeable alkaloid discovered in Apocyanaceae plants (Dalton et al., 1967). It was observed that EPED3 at nanomolar concentrations exhibited an extraordinary ability to kill all tested myeloma cell lines, including those sensitive to dexamethasone, etoposide (VP- 16-213) and doxorubicin.
  • the present invention also investigated the mechanism of EPED3's cytotoxic effects on myeloma cells.
  • ellipticine was reported binding to nucleic acids and acts as an inhibitor of topoisomerase-II (topo II) to stimulate topo II- mediated DNA breakage
  • topo II topo II
  • the results presented herein indicated that EPED3 directly impacted cytoplasmic organelles, particularly targeting mitochondria, which subsequently triggered formation of ap ⁇ ptosomes and sequential activation of the cell death cascade.
  • This mechanism was consistent with the ability of ellipticine to uncouple mitochondrial oxidative phosphorylation, presumably through its accumulation within the inner mitochondrial membrane and subsequent inhibition of the electron pathway of cytochrome c oxidase (Sureau et al., 1993).
  • Other groups however, have suggested different potential mechanisms for EPED3. For instance, it was implied that EPED3 was an inhibitor of RNA synthesis or an inducer of endoplasmic reticulum stress.
  • the present invention was directed to developing agents for treating multiple myeloma
  • the in vitro experiments were conducted in clinically relevant settings.
  • the disease relies on dynamic interactions — both direct and indirect — between myeloma cells and bone marrow-derived stromal cells, and this synergy can efficiently protect malignant cells from drug-induced apoptosis (Nefedova et al., 2003).
  • the in vitro EPED3 cytotoxicity studies were conducted on co-cultured myeloma and stromal cells to mimic the clinical setting and provide a protective environment to myeloma cells.
  • the mesenchymal cells derived from human fetal bone were cultured with myeloma cells, both with and without direct contact between cell types.
  • nanomolar concentrations of EPED3 resulted in rapid cell cycle arrest and massive apoptosis in myeloma cells.
  • the present invention has also demonstrated the toxic effect of EPED3 on cancer cells other than myeloma cells.
  • EPED3 is emerging as a novel agent in future tailored cancer treatments for individual patients' drug resistances.
  • a metthod of treating myeloma in an individual comprising administering a pharmacologically effective dose of ellipticine, a derivative thereof or a combination thereof to the individual.
  • the ellipticine or the derivative thus administered may induce cell cycle arrest of myeloma cell, may induce apoptosis of myeloma cell, may overcome acquired drug resistance or a combination thereof without affecting the viability of normal cells.
  • Examples of such derivatives of Ellipticine may include but are not limited to EPED3 (9-dimethyl amino-ethoxy elipticine), or NSC69187 (9-methoxy ellipticine).
  • the type of individual that may benefit from such a method may be the one diagnosed with myeloma or the one resistant to drugs such as doxorubicin.
  • the route of administration of ellipticine, its derivative or a combination thereof may include but is not limited to oral, topical, intraocular, intranasal, parenteral, intravenousm intramuscular or subcutaneous route.
  • the dose range of the administered ellipticine, the derivative of ellipticine or its combination may be from about 0.01 mg/kg to about 500 mg/kg body weight of the individual.
  • a method of treating myeloma in an individual comprising administering a pharmacologically effective dose of a topoisomerase II inhibitor, wherein said inhibitor induces cell cycle arrest of myeloma cell, induces apoptosis of myeloma cell, overcomes acquired drug resistance or a combination thereof without affecting the viability of normal cells, thereby treating myeloma in the individual.
  • the topoisomerase inhibitor used in such a method may be ellipticine or its derivative.
  • the examples of the ellipticine derivatives, the dose administered, route of administration and the type of individual benefitting from such a method is the same as discussed supra.
  • a method of inhibiting growth of a myeloma cell comprising: contacting the myeloma cell with ellipticine, a derivative thereof or a combination thereof. Such a contact may inhibit myeloma cell growth by inducing cell cycle arrest, apoptosis or a combination thereof. Specifically, the apoptosis may be induced by activation of caspase 9. Additionally, the examples of derivatives that may be used in such a method is the same as discussed supra. Furthermore, the examples of myeloma cell may include but are not limited to ARPl, CAG, L363, MM 144, OCI-my5, OPM2 or U266.
  • a method of inducing apoptosis of a myeloma cell comprising contacting the myeloma cell with ellipticine, a derivative thereof or a combination thereof such that the contact activates caspase-9, thereby inducing apoptosis of the myeloma cell.
  • the term, "a” or “an” may mean one or more.
  • an may mean one or more than one.
  • another or “other” may mean at least a second or more of the same or different claim element or components thereof.
  • the term “contacting” refers to any suitable method of bringing the sample into contact with the ellipticine, its derivative or a combination thereof. In vifro or ex vivo may be achieved by exposing the above-mentioned cell to the composition in a suitable medium.
  • compositions described herein can be administered independently, either systemically or locally, by any method standard in the art.
  • Dosage formulations of the composition described herein may comprise conventional non-toxic, physiologically or pharmaceutically acceptable carriers or vehicles suitable for the method of administration and are well known to an individual having ordinary skill in this art.
  • composition described herein may be administered independently or in combination with any other anti-neoplastic or chemotherapeutic agent and may comprise one or more administrations to achieve, maintain or improve upon a therapeutic effect. It is well within the skill of an artisan to determine dosage or whether a suitable dosage of the composition comprises a single administered dose or multiple administered doses. An appropriate dosage depends on the subject's health, the inhibition of myeloma cell growth either by inducing cell cycle arrest or by inducing apoptosis, the route of administration and the formulation used.
  • Cell Titer-Glo luminescent cell viability assay kit from Promega Co (Madison, WI) was used to determine cell growth inhibition by the various compounds tested. The kit determines cell viability by quantifying the amount of ATP present in the cell culture medium. Presence of ATP signals the presence of metabolically active cells (3). The intensity of luminescence was measured by a computerized luminometer (Promega, Co. Madison, WI). Each well was read five times within 30 minutes of adding Cell Titer-Glo reagent mixture.
  • Cells were contacted with the antineoplastic agents in ninety six well culture plates. The concentration of each neoplastic agent was maintained a constant. A cell culture medium control and 0.5% DMSO control were maintained in each set of experiments. The cell viability was tested every day using the CellTiter-Glo luminescent cell viability kit from Promega Co. (Madison, WI) for a period of five days.
  • the CellTiter-Glo Luminscent Assay showed no significant difference in cell viability in the control (medium alone) and medium + DMSO (5%). Additionally, of the 5 compounds that were tested, NSC 338258 showed a significant anti-myeloma effect on all 4 myeloma cell lines ( Figures 1, 2, 3, 4). Furthermore, this effect was more than other compounds that were tested
  • Myeloma cell lines were exposed to NSC 338258 and existing ami myeloma drugs, Adriamycin® (Doxorubicin Hydrochloride) and Etoposide (VP- 16-213) in ninety six well culture plates. The concentration of each compound was set at 0.2 mM. The cell viability was tested every day using the CellTiter-Glo luminescent cell viability kit from Promega Co. (Madison, WI) for a period of five days. In vitro results showed that NSC 338258 had immediate inhibition of cell proliferation within the first 24 hours. The cell growth was near zero in the following days (day 4 onwards; Figure 6).
  • NSC 338258 Fetal bone mesenchymal cells were contacted with NSC 338258 in ninety six well culture plates. The concentrations of NSC 338258 were set at 0.01, 0.03. 0.1, 0.3 and 1.0 ⁇ M ( Figure 7). The cell viability was tested every day using the CellTiter-Glo luminescent cell viability kit from Promega Co. (Madison, WI) for a period of five days.
  • NSC 338258 To test the overall cell growth inhibition of NSC 338258, twelve non myeloma cancer cell lines, HEL, HL-60, k562, MEGOl, THPl, HeIa, G401, Dul45, sw480, sw620, saoS2, and MG63, were exposed to the compound in 96 well cell culture plates for a period of five days. A cell culture medium control and 0.5% DMSO control were maintained in each set of experiments. The cell viability was tested every day using the CellTiter-Glo luminescent cell viability kit from Promega Co. (Madison, WI) for a period of five days (figures 8A-8B). EXAMPLE 9
  • Human myeloma cell lines (OPM-2, RPMI 8226, and U266) were purchased from the American Type Culture Collection, Manassas, VA ). Except ARK, ARPl, and CAG myeloma cell lines, most of the myeloma cell lines were provided by Michael Kuehl,
  • the RPMI 8226/Doxl V drug-resistant variant was the gift of William S. Dalton (H. Lee
  • All cell lines were maintained in RPMI- 1640 medium supplemented with 10% FBS, lOOunit/ml of penicillin/streptomycin, 2mM of L-glutamine, and ImM of sodium pyruvate (Invitrogen Co, Carlsbad, CA). Cells were incubated at 37°C with 5% CO 2 . Cell viability was determined using hemacytometer with trypan blue stain (Invitrogen, Co, Carlsbad, CA).
  • VP-16-213 were purchased from Sigma (St. Louis, MO). Velcade (bortezomib or PS-341) was provided by Millennium Pharmaceuticals Inc (Cambridge, MA). Dexamethasone was purchased from Elkins-Sinn (Cherry Hill, NJ).
  • the monolayer human fetal bone mesenchymal cells were established from live bone chips (Advanced Bioscience Resources Inc, Alameda, CA) in complete RPMI- 1640 medium. The medium was the same as described above. The cells were trypsinized and seeded in 6-well plates, and then allowed to reach 75% confluence.
  • myeloma cells were suspended in the TC culture inserts with 3.0- ⁇ m pore size track-etched polyethylene terephthalate (PET) membranes (Becton Dickson Labware, Franklin Lakes, NJ) at 10 6 cells/ml of total volume of medium (10 ml/well), while the human fetal bone mesenchymal cells were adhering to the bottom of the plates. Anti- myeloma agents were added individually at desired concentrations.
  • PET polyethylene terephthalate
  • human mesenchymal cells were seeded in 96- well plates with 50 ⁇ l of medium, and then allowed to reach 50% confluence.
  • Myeloma cells (25- ⁇ l aliquot) were added to wells with mesenchymal cells to reach a final concentration of 5,000 myeloma cells/well.
  • 75 ⁇ l of medium contains non- drug, NSC 338258 (EPED3), or Velcade at desired concentrations.
  • Myeloma cell lines were maintained at >90% viability at 37°C and 5% CO 2 .
  • Cells were diluted to 20,000 cells/ml in fresh medium.
  • a 50- ⁇ l aliquot of cell suspension was seeded in 96-well tissue culture plates to final cell counts of 1,000 cells/well.
  • AU compounds obtained from NCI's Developmental Therapeutics Program (DTP compounds) were dissolved in 100% dimethylsulfoxide (DMSO) (Sigma- Aldrich, St. Louis, MO) at 1 mg/ml and further diluted in fresh medium to 1 ⁇ g/ml.
  • DMSO dimethylsulfoxide
  • Each compound (50- ⁇ l aliquots) was distributed to a well containing 1,000 myeloma cells.
  • Each plate included controls with addition of only medium or only DMSO (0.5% final concentration). All experiments were conducted in triplicate.
  • CellTiter-Glo Luminescent Assay kit Promega, Madison, WI
  • Myeloma cells were suspended in fresh medium at 2X10 5 cells/ml and seeded into 96-well plates in 50- ⁇ l aliquots (10,000 cells/well). After treating cells in each well with the appropriate compound (50- ⁇ l aliquots of 2X stock solution) and medium control, the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay kit (Promega, Madison, WI) was used to determine cell proliferation, according to manufacturer's instructions. Briefly, dye solution was added to each well (15 ⁇ l/well) and incubated for 4 hours at 37°C and 5% CO 2 ; 100 ⁇ l of the solubilization solution/stop mix was then added. The plates were read at 570 nm in a plate reader, and the A570 nm values were corrected by A650 nm. All experiments were conducted in triplicate.
  • MTT methylthiazolyldiphenyl-tetrazolium bromide
  • the 8226/DoxlV cell line was established by chronic exposure to Dox in presence of P-glycoprotein inhibitor, verapamil and was characterized by reduced expression and activity of topo II (Futscher et al., 1993).
  • the cell line demonstrated cross- resistance to other topo II poisons such as mitoxantrone and etoposide, but was not resistant to chemotherapeutic agents not known to target topo II, including melphalan, vincristine, cytarabine, or dexamethasone.
  • the 8226/DoxlV and its parental line, RPMI 8226, cells were plated in 96- well plates at 10,000 cells/well and incubated with serial dilutions of EPED 3 or Dox, as previously described (Landowski et al., 1999). After 96 hours incubation at 37°C, MTT was added to each well at a final concentration of 20 ⁇ g/ml and incubated an additional 4 hours. Plates were centrifuged and the media replaced with 0.1 ml DMSO to solublize the formazan complex. Optical density was measured at 540 nm using a Bio-Tek microplate spectrophotometer. IC50 values were calculated by linear regression analysis (Origins® Software Technologies, Inc.) of the log-linear plot for percent survival versus log drug concentration.
  • the GeneChip Human Genome U133Plus 2.0 Array system (Affymetrix, Inc., Santa Clara, CA) was utilized to conduct comprehensive analysis of genome-wide expression profiles in primary myeloma cells.
  • the erythrocyte-depleted fraction of bone marrow aspirates were sorted for CD138 + myeloma plasma cells using the AutoMACS magnetic cell separating technique (Miltenyi Biotec, Auburn, CA).
  • the previously described cDNA microarray standard protocol was used (Zhan et al., 2003).
  • Cell cycle arrest was measured using DNA-Prep Coulter Reagents kit (Beckman Coulter, Inc., Miami, FL). Myeloma cells were harvested from co-culture inserts and washed in phosphate buffered saline (PBS); 5 XlO 5 cells were stained according to the manufacturer's instructions. Samples were analyzed using EPICS XL-MCL flowcytometer equipped with EXPO32 ADC software (Beckman Coulter, Inc.). Apoptosis was detected using the Annexin V-fluorescein isothiocyanate (FITC)/7-AAD kit (Beckman Coulter, Inc.).
  • FITC Annexin V-fluorescein isothiocyanate
  • Myeloma cells were harvested from co-culture inserts and washed in PBS; 5xlO 5 cells were incubated on ice with 100 ⁇ l of binding buffer (10 ⁇ l Annexin V-FITC and 20 ⁇ l of 7-AAD) for 15 minutes. Samples were analyzed using EPICS XL-MCL flowcytometer equipped with EXPO32 ADC software (Beckman Coulter, Inc.).
  • Myeloma cells were co-cultured with human fetal bone mesenchymal cells in the TC inserts. The cells were treated with individual antimyeloma agents at desired concentrations. An aliquot containing 10 6 cells was transferred to 24- well plate and mixed with MitoTracker Red CMXRo s (Invitrogen) at 5OnM final concentration. After a 15- minute incubation, cells were collected and washed with PBS and resuspended at 5X10 5 cells/ml for slide adhesion by the Cytospinning method.
  • the slides were fixed in 3.7% formaldehyde/PBS for 15 minutes and rinsed in PBS; cells were permeabilized in ice-cold acetone for 5 minutes and rinsed in PBD (PBS with 0.1% NP-40) solution twice.
  • Monoclonal mouse anti-human cytochrome c antibody (R&D Systems, Minneapolis, MN) was diluted (10 niM Na + PO 4 [pH 7.8], 0.15 M NaCl) 1:500 and added to each spot. After 15 minutes, slides were washed twice in PBD.
  • the FITC-conjugated goat anti-mouse IgG H+L chain (GAM) (BD Biosciences, San Diago, CA) was added, and slides were incubated for 15 minutes.
  • Myeloma cell lines were indirectly co-cultured with human mesenchymal cells as described above. EPED3 and Velcade were added to co-culture to final concentrations of 2 ⁇ M and 0.5 ⁇ M, respectively. After 6 hours of treatment, myeloma cells were harvested from the inserts, washed twice in PBS, and resuspended in protein extraction buffer (PBS, 10 ⁇ g/ml aprotinin, 10 ⁇ g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) to a final concentration of 10 4 cells/ ⁇ l. Cell Iy sates were prepared by three cycles of freezing in liquid nitrogen followed by thawing at 37 0 C.
  • PBS protein extraction buffer
  • Protein concentration was determined by spectrophotometry (NanoDrop Technologies, Wilmington, DE). For sodium dodecyl-polyacrylamide gel elctrophoresis (SDS-PAGE), 100 ⁇ g of protein extract was mixed with NuPAGE LDS sample buffer (Invitrogen) and loaded into each lane of the gel (10%— 15% polyacrylamide separating gel, 4% stacking gel). After electrophoresis, protein was transferred to Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The primary antibodies (goat anti-human caspase-3, -8, and -9 polyclonal antibodies) were (R&D Systems) detected with WesternBreeze kit (Invitrogen).
  • SDS-PAGE sodium dodecyl-polyacrylamide gel elctrophoresis
  • EPED3 demonstrated significant killing activities among all tested DTP compounds, and this antimveloma efficacy cannot be prevented by stromal cells in the co-culture system
  • Amplification and overexpression of CKSlB has been linked with de novo high-risk myeloma (Shaughnessy, 2005), and this putative oncogene maps to chromosome arm Iq21, which has high frequency of genomic instability in multiple myeloma (Sawyer et al., 2005).
  • the COMPARE algorithm was used to query growth inhibition data in DTP's open database of approximately 44,000 synthetic compounds to identify potential anticancer compounds with higher potency in cells with increased expression of CKSlB.
  • GI50 values the concentration that causes 50% growth inhibition — were used as selection criteria for this analysis.
  • NSC 338258 EPED3
  • NSC 69187 a compound that inhibited cell viability in four myeloma cell lines. Both these agents are derivatives of ellipticine. The ellipticine and their derivatives were examined further in myeloma-growth inhibition.
  • Myeloma cells were also co-cultured with human fetal bone mesenchymal cells. JJN3, L363, OPM2, and U266 cells were exposed to EPED3 (2 ⁇ M) or Velcade (0.5 ⁇ M) in co-culture without direct contact with stromal cells. Cell viability was assayed every 24 hours with CellTiter-Glo reagent in following 6 days. Myeloma cell destruction was observed with both EPED3 and Velcade treatment; linear regression analysis indicted that the anti-myeloma efficacy of EPED3 highly significant associated with Velcade- treatment, ( ⁇ 0.98, /KCOOOl; Table 3) in all four myeloma cell lines tested. Importantly, however, severe destruction of the mesenchymal cells was observed under Velcade treatment, while such morphological damage was not seen under EPED3 treatment (Figure 11). Table 3
  • EPED3 is a highly effective antimyeloma agent that inhibits viability and overcomes acquired drug resistance
  • U266 cells were exposed to EPED3 (2 ⁇ M) or Velcade (0.5 ⁇ M) in co- culture without direct contact with fetal human bone mesenchymal cells. Twelve hours after treatment, cells were harvested for using flow cytometry to detect ongoing apoptosis and cell cycle arrest.
  • the cell cycle arrest analysis was simultaneously performed to examine DNA helix in U266 cells at 12 hours. It was observed that 67.5% of the EPED3 -treated population was in Go+Gt phase as opposed to 49.5% of the control population and 59.8% of Velcade-treated populations in Go+Gt phase. Furthermore, 7.2% of the EPED3-treated population was in G 2 +M phase as opposed to 25.4% of the control and 18.9% of Velcade- treated populations in the G 2 +M phase ( Figure 14A- 14C).
  • caspases involved the mitochondrial apoptotic pathway was also examined using Western blotting. JJN3, L363, OPM2, and U266 cells were co- cultured with human fetal bone mesenchymal cells without contact and treated with EPED3 or Velcade for 6 hours. Proteins were then extracted from the harvested cells and analyzed for activation of caspase-8, -9, and -3 ( Figure 15C). There was no obvious activation of caspase-8 in any myeloma cell line tested, which excluded the involvement of the extrinsic apoptotic pathway. On other hand, there was a strong indication of activation of caspase-9 in all myeloma cell lines. Furthermore, it was observed that caspase-3 was cleaved in treated cells ( Figure 15C), further indicating an activated intrinsic apoptotic process in triggering the proteolytic cascade.
  • NSC 338258 or EPED3 is prepared using a patented 5-step process. This provides 1 gm of the 9-OH metabolite and enough additional 9-OH to make the glucuronide and the sulfate by enzymatic synthesis. Additionally, [ 14 C] EPED3 is also synthesized.
  • a reverse- phase HPLC method is used to separate the parent compound and the three metabolite standards to be synthesized. It utilizes an Agilent Model 1100 or 1050 HPLC equipped with a UV and Radiomatic detector for the visualization of unlabeled and radiolabeled compounds, respectively.
  • Excreta collection vessels are maintained at ⁇ 4°C (ice-water bath). Concentration and percent of administered radioactivity are determined by direct liquid scintillation counting of urine aliquots and CO 2 trapping solution (10% KOH) aliquots and by combustion of feces followed by LSC. HPLC analysis of metabolites are performed directly on urine samples. Urine from all 5 treated animals are pooled for each collection period.
  • Pharmacokinetics and Tissue distribution of the compound (intravenous administration, 6mg/Kg, I X lO 6 DPM/mouse): 3 female SCID bg mice per time point are serially sacrificed for blood samples obtained by heart puncture and placed in heparinized tubes.
  • mice are acclimated to caging and feed for 1 week prior to dosing.
  • the mice are provided with feed and water ad libitum throughout the study period.
  • the blood samples are collected at 0, 5, 10, 20, 30, 45, 60, 120, 180, 240 minutes and 24 hours.
  • the concentration is determined by combustion of 50 _L of whole blood followed by direct liquid scintillation counting (LSC).
  • LSC direct liquid scintillation counting
  • HPLC analysis is performed on 0, 10, 30, 60, 120, 240-minute and 24- hour samples (all 3 animals blood pooled for analysis) to determine the metabolic profile.
  • the following tissues are removed from the 60 and 240-minute animals for analysis of total 14 C concentration (EPED3 equivalents) by combustion analysis and subsequent LSC: liver, kidneys, brain, heart, lungs, salivary gland, pancreas, adrenals, thyroid, thymus, lymph nodes, spleen, bone marrow, adipose, skeletal muscle, stomach, small intestine and large intestine. Contents are removed from intestines prior to analysis.
  • Biliary excretion of [ 14 C] EPED3 i.v. administration 6mg/Kg, 5 X 10 6 DPM/rat
  • metabolites by rats 5 female Sprague-Dawley rats with canulated bile ducts are used for bile sample collection. Their carotid arteries are canulated for dose administration. The rats are acclimated to caging and feed for 1 week prior to dosing. The rats are provided with feed and water ad libitum throughout the study period. The samples are collected at 0, 5, 10, 20, 30, 45, 60, 120, 180, 240 minutes and 24 hours. At time 0, the rat will serve as the control.
  • the present invention also examines novel therapeutic approaches for myeloma using experimental animal models.
  • the anti-myeloma efficacy of 9- (Dimethylaminoethoxy)-ellipticine is examined using a panel of myeloma cell lines engrafted in severe combined immunodeficient (SCID) mice.
  • SCID severe combined immunodeficient
  • the 3 cell lines are infected with a lentivirus-containing luciferase. Luciferase-expressing myeloma cells can be traced in live animals using the Xenogen IVIS 200 luminescence imaging system.
  • Six to 8 weeks old SCID mice are subcutaneously injected with 10 7 myeloma cells.
  • Tumor growth in live animals is monitored by measuring circulating level of monoclonal human immunoglobulins (hlg) in mice sera using ELISA (Yaccoby et ah, 1998; Yang et ah, 2002) and through imaging of tumor cells luminescence intensity.
  • the tumor growth is detected within one week by ELISA and within 1-3 weeks by imaging.
  • mice Upon establishment of myeloma growth as determined by both methods, mice are randomly divided into 3 groups; one control and 2 treated with different doses of 9-(Dimethylaminoethoxy)-ellipticine (8 mice/group for each cell line). Mice are implanted with Alzet osmotic pumps delivering 0.25 ⁇ l of drug solution per hour for 28 days. The daily doses are 25 and 50 mg/kg/day. Control animals have pumps loaded with buffer (PBS) only. At the experiments' end, mice are subjected to imaging analysis, bled and sacrificed. Subcutaneous tumors are removed, weighed, histologically processed and internal organs are examined for potential damage. Through out the experimental period the mice are weighed and closely observed for potential side effects. It is contemplated that 9- (Dimethylaminoethoxy)-ellipticine effectively inhibits myeloma cell growth in vivo with no or minimal side effects.
  • PBS buffer

Abstract

La présente invention porte sur l'inhibition sélective de la croissance cellulaire de cellules myélomateuses par des dérivés de l'ellipticine, la 9-méthoxy ellipticine et la 9-diméthyl amino-éthoxy ellipticine. Parmi les dérivés de l'ellipticine testés, la 9-diméthyl amino-éthoxy ellipticine s'est révélée être la plus efficace pour inhiber la croissance cellulaire. La toxicité cellulaire de la 9-diméthyl amino-éthoxy ellipticine s'est montrée sélective pour les cellules myélomateuses et n'a pas tué de cellules normales dans la marge posologique antinéoplasique effective. La 9-diméthyl amino-éthoxy ellipticine s'est avérée plus efficace que des médicaments antimyélomes existants, tels que l'Adriamycine® et l'Etoposide, pour provoquer une réponse rapide et meilleure d'inhibition de la croissance cellulaire.
PCT/US2006/029212 2005-07-27 2006-07-27 Activites antineoplasiques de l'ellipticine et de ses derives WO2007016254A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US70294405P 2005-07-27 2005-07-27
US60/702,944 2005-07-27

Publications (2)

Publication Number Publication Date
WO2007016254A2 true WO2007016254A2 (fr) 2007-02-08
WO2007016254A3 WO2007016254A3 (fr) 2009-04-30

Family

ID=37709173

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2006/029212 WO2007016254A2 (fr) 2005-07-27 2006-07-27 Activites antineoplasiques de l'ellipticine et de ses derives

Country Status (2)

Country Link
US (1) US20070027175A1 (fr)
WO (1) WO2007016254A2 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004022580A2 (fr) 2002-09-09 2004-03-18 Dana-Farber Cancer Institute, Inc. Peptides bh3 et leur methode d'utilisation
CA2645853A1 (fr) 2006-03-31 2007-11-01 Dana-Farber Cancer Institute Procedes de determination de la chimiosensibilite cellulaire
US20080187930A1 (en) * 2006-11-07 2008-08-07 Shaughnessy John D Gene expression profiling based identification of genomic signature of high-risk multiple myeloma and uses thereof
US20080274911A1 (en) * 2006-11-07 2008-11-06 Burington Bart E Gene expression profiling based identification of genomic signature of high-risk multiple myeloma and uses thereof
US8513274B2 (en) * 2009-03-17 2013-08-20 Etzem, Inc. Ellipticine compounds for treating obesity
WO2014047342A1 (fr) 2012-09-19 2014-03-27 Dana-Farber Cancer Institute, Inc. Profilage dynamique du bh3
CA2922503C (fr) * 2013-09-19 2021-10-26 Dana-Farber Cancer Institute, Inc. Procede de profilage de bh3
EP3288964B1 (fr) 2015-04-27 2024-02-21 Dana-Farber Cancer Institute, Inc. Profilage bh3 à haut rendement : une technologie rapide et échelonnable pour un profil bh3 sur un faible nombre de cellules
CN104830776A (zh) * 2015-05-05 2015-08-12 中国医学科学院血液病医院（血液学研究所） 一种耐药型多发性骨髓瘤动物模型及其构建方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4310667A (en) * 1976-04-22 1982-01-12 Agence Nationale De Valorisation De La Recherche (Anvar) 2-N Quaternary ammonium salt derivatives of 9-hydroxy ellipticine
WO2004096224A2 (fr) * 2003-04-29 2004-11-11 Boehringer Ingelheim International Gmbh Combinaisons pour traiter des maladies impliquant la proliferation cellulaire, la migration ou l'apoptose de cellules du myelome ou l'angiogenese

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4310667A (en) * 1976-04-22 1982-01-12 Agence Nationale De Valorisation De La Recherche (Anvar) 2-N Quaternary ammonium salt derivatives of 9-hydroxy ellipticine
WO2004096224A2 (fr) * 2003-04-29 2004-11-11 Boehringer Ingelheim International Gmbh Combinaisons pour traiter des maladies impliquant la proliferation cellulaire, la migration ou l'apoptose de cellules du myelome ou l'angiogenese

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SVOBODA G.H. ET AL.: 'Alkaloids of Ochrosia maculata Jacq. (Ochrosia borbonica Gmel.): Isolation of the Alkaloids and Study of the Anitumor Properties of 9-Methoxyellipticine' JOURNAL OF PHARMACEUTICAL SCIENCES vol. 57, no. 10, 1968, pages 1720 - 25 *

Also Published As

Publication number Publication date
WO2007016254A3 (fr) 2009-04-30
US20070027175A1 (en) 2007-02-01

Similar Documents

Publication Publication Date Title
US20070027175A1 (en) Antineoplastic activities of ellipticine and its derivatives
RU2757373C2 (ru) Комбинированная терапия противоопухолевым алкалоидом
Weber et al. DNA damaging drugs
US6403563B1 (en) Antitumor composition containing a synergistic combination of an anthracycline derivative with a camptothecin derivate
ES2282400T3 (es) Composiciones antitumorales que contienen derivados de taxano.
US6537990B1 (en) Combined preparations comprising morpholine anthracyclines and anticancer agent
RU2391101C2 (ru) Комбинированное применение эктеинасцидина-743 и содержащих платину противоопухолевых соединений
EP3157336A1 (fr) Oxabicycloheptanes et oxabicycloheptènes pour le traitement du cancer des ovaires
US20120202840A1 (en) Use of Flubendazole and Vinca Alkaloids for Treatment of Hematological Diseases
TWI430801B (zh) 使用小檗鹼化合物製造抑制癌幹細胞生長或轉移的藥劑之用途
US20050119337A1 (en) Methods of application of Schisandrin B in the preparation of anticancer medications
US9492469B2 (en) Combination therapy for the treatment of proliferative diseases
Potmesil et al. Preclinical and clinical development of DNA topoisomerase I inhibitors in the United States
Gamucci et al. Activity and toxicity of GI147211 in breast, colorectal and non-small-cell lung cancer patients: An EORTC—ECSG phase II clinical study
US10765675B2 (en) Compositions and methods for treating Ewing family tumors
AU4338493A (en) Treatment of cancers
CN1327842C (zh) 一种新型的抗肿瘤药物复方制备方法
CA2557857C (fr) Traitement contre le cancer au moyen de l'inhibiteur de la topoisomerase-ii, un bis-dioxypiperazine et par radiotherapie
KR20100107461A (ko) 의약 조성물 또는 조합제
ElBagoury et al. Chemotherapy over the years
CN111773388A (zh) A-失碳-5α雄甾烷化合物类药物与抗癌药物的联合应用
EP0969839B1 (fr) Anhydrovinblastine pour le traitement du cancer
WO2022093919A1 (fr) Inhibiteurs d'exo1 en association avec d'autres médicaments anticancéreux pour inhiber la croissance de cellules cancéreuses
WO2022093789A1 (fr) Inhibiteurs à petites molécules d'apex, seuls et en combinaison avec d'autres médicaments contre le cancer pour inhiber la croissance de cellules cancéreuses
TWI406658B (zh) 使用異硫氰酸酯類來治療癌症之方法及用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06788666

Country of ref document: EP

Kind code of ref document: A2